Human immunodeficiency virus 1 (HIV-1) infection is a slowly replicating retrovirus that leads to acquired immunodeficiency syndrome (AIDS). In this paper, the PDB file of gp120 and CD4 were obtained from the Protein Data Bank. The Expasy-ProtParam tool was used to analyze the physicochemical properties of HIV-1 gp120. ClusPro 2.0 was used to dock the interaction between gp120 and CD4. We described structural basis, affinity and kinetics of binding, and explored how CD4 cumulatively targeted most of the external surface of the envelope glycoprotein. These results showed that uncleaved gp120 had native-like structures and supported their used as candidate antibodies.

• HIV-1 gp120 • CD4 Receptor • Conformational Changes

Long Y, Liang H, Long Z, Wang P, Xie X, et al. (2026) CD4 Binding Induces Conformational Changes in the Gp120 Glycoprotein. Ann Med Chem Res 8(1): 1027.

The HIV envelope glycoprotein gp120 subunit interacts with CD4, triggering a series of conformational changes. Gp120 contains a CD4 binding site, several variable regions (V1-V5) and a conserved core region variable region forming an exposed ring structure responsible for recognizing and binding to receptors on the host cell surface. But also create the conditions necessary for the activation of gp41 [1-3]. In conclusion, when the trimeric HIV-1 Env binds to the cell surface receptor CD4, HIV-1 begins to infect CD4+ T cells. Binding of CD4 induces conformational changes within the gp120 subunit of Env that allow subsequent binding of the co-receptors CCR5 or CXCR4. Receptor and co-receptor binding induce conformational changes in the Env gp41 subunits. These changes drive fusion of the virus and host cell membranes [4,5]. Therefore, the analysis of the molecular mechanism between HIV-1 envelope glycoprotein and host cell CD4 receptor probably has consequences for antibody mediated immune responses and vaccine design.

Proteins play an important role in the process of life through conformational changes. The essence is to allow interacting proteins to perform specific biological functions, such as intercellular communication and gene expression [6-9]. In other words, the function of proteins is affected by protein conformation and protein-protein interaction [10]. Therefore, the study of protein-protein interactions will help to discover more protein functions and explain molecular mechanisms. Due to the complexity of determining protein-protein interaction sites based on experiments, docking tools based on network and computer algorithms have been continuously developed and improved over the past 15 years. For example, ZDOCK uses the Fast Fourier Transform algorithm to achieve efficient global docking search on a 3D grid while scoring using a combination of shape complementarity, electrostatics, and statistical potential terms [11]; HADOCK is driven by experimental data [12]; GRAMM-X uses smoothed potentials, refinement stages, and knowledge based scoring based on the Fast Fourier Transformation methodology [13]; and ClusPro 2.0 uses physical principles and algorithms to simulate and optimize the conformational space of protein-protein interactions to find the most likely docking conformation [14]. In addition to the above docking tools, it has recently been reported that artificial intelligence-based prediction methods will further improve the accuracy of predicting protein-protein interactions [15]. It is the development of bioinformatics that provides convenience for revealing the interaction between biological macromolecules [16-19]. In order to investigate the molecular mechanism of interaction between HIV-1 and host cells, we used ClusPro 2.0 to dock gp120 and CD4, which is a fully automated protein-protein docking tool with efficient, accurate, and reliable features and advantages. PyMoL visualized the binding sites and analyze the interacting amino acid residues. Meanwhile, we performed sequence alignment and phylogenetic tree construction of HIV-1 env amino acid sequences from FM865453-FM865531 in Hainan Province, which were obtained from EMBL.

gp120 and CD4 sequences retrieval

Gp120 PDB file was found from AlphaFold Protein Structure Database, which is clade A/E 93TH057 HIV-1 gp120 core. CD4 PDB file 1CDH were obtained by the Protein Data Bank [19].

Prediction of physicochemical properties

ProtParam online software was provided by ExPASy proteomics tools, which could predict certain properties about a protein. Hence, we used the tool to predict the properties of gp120 and CD4, which included molecular composition, molecular weight, theoretical isoelectric point, and instability index. Meanwhile, we predict the amino acid distribution and charge of gp120.

Sequence alignment and Construct Maximun Likelihood Tree

A phylogenetic tree depicts the evolutionary history of a given set of species. phylogenetic tree estimation method consists of four major steps. First, the set of k-mers present in each input sequence is generated. Second, a binary matrix is constructed. Third, a suitable value of k is chosen based on entropy values. Finally, a phylogenetic tree is constructed using maximum likelihood estimation.

Computational modeling of gp120-CD4 complex structures

In order to determine the binding sites and binding strength between the gp120 and CD4, we utilized ClusPro 4.0 to predict structure of protein-protein complex interactions [14-22]. In addition, PyMOL was used to dock complexes and possess the ability to visualize the binding sites of complexes and calculate the strength of binding sites.

Physicochemical properties of gp120 and CD4

The gp120 sequence starts with the N-terminal Val (V) and has a total of 353 amino acids. The calculated molecular weight was 39168.53. The theoretical isoelectric point (PI) of the gp120 sequence is 6.84, that is, the pH of the protein without net charge. The total number of negatively charged residues (Asp + Glu) was 34, while the total number of positively charged residues (Arg + Lys) was 33. The total number of atoms in the protein molecule is 5438, the molecular formula is C1714H2696N478O527S23, the instability index is 39.09, the aliphatic index is 76.46, and the total average hydrophobicity (GRAVY) is -0.403 (Table 1).

Table 1: Physicochemical properties of gp120 core and CD4

The CD4 sequence consists of 178 amino acids with a molecular weight of 19,700.44 Da and a theoretical isoelectric point (pI) of 8.88. It contains a total of 19 negatively charged residues (Asp + Glu) and 23 positively charged residues (Arg + Lys). The molecular formula of the protein is C865H1414N242O273S4, comprising a total of 2,798 atoms. Based on the calculated instability index (II) of 44.66, this protein is classified as unstable. Additionally, the aliphatic index is 89.72, suggesting a relatively high thermostability, while the grand average of hydropathicity (GRAVY) value is -0.480, indicating that the protein is overall hydrophilic in nature.

Maximun Likelihood Tree

The ML tree revealed well-supported phylogenetic clusters corresponding to known HIV-1 subtypes and circulating recombinant forms (CRFs), including CRF01_ AE and CRF07_BC. Distinct color-coded clades indicated clear genetic differentiation and potential transmission clusters. Sequences from the same epidemic lineage clustered closely, reflecting recent common ancestry. Sequence alignment ((Figure 1a and 1b), identified multiple hypervariable sites, highlighted in red, mainly located in genomic regions encoding envelope proteins.

Figure 1 a, Maximum Likelihood Phylogenetic Tree Circular tree showing genetic relationships of viral sequences. Colored clades represent distinct subtypes/ transmission clusters. Scale bar (0.1) indicates genetic distance. b, Multiple Sequence Alignment Aligned viral sequences with positions of high variability highlighted in red. Letters denote nucleotides (A/T/C/G), and - indicates gaps. Top numbering shows alignment position.

These variable regions are closely associated with viral immune evasion and host adaptation. In contrast, relatively conserved regions were observed in core structural and enzymatic genes (e.g., gag, pol), which are essential for viral replication and function.

gp120-CD4 interaction

Through ClusPro 2.0: protein-protein docking, we obtained 10 display models. The polarity of 10 display models was analyzed using PyMoL. The interaction of amino acid residues between CD4 and gp120 was represented by hydrogen bonds. Eight display models are shown (Figure 2).

Figure 2 The docking models of gp120 and CD4. a, display models 1. Gp120 and gp41 amino acid residues interact through hydrogen bonds. b, display models 2 c, display models 3. d, display models 4. e, display models 5. f, display models 6. g, display models 7. h, display models 9.

The amino acid residues that interact with CD4 in these 10 display models include GLN-39, Ala 105, ASP-35, THR-28, HIS-18, CYS-31, THR-8, GLN-39, ASP-64, ALA-17, ALA-27, HIS-29, PRO-33, GLU-44.

Technological advances have taken a significant role in protein structure prediction. The AlphaFold server can accurately predict the structure of proteins through amino acid sequences, providing a path different from the laboratory for analyzing protein structure. We obtained the HIV-1 gp120 core PDB file from the AlphaFold Protein Structure Database and analyzed the physicochemical properties of its amino acid sequence. We found that the gp120 amino acid sequence was weakly negatively charged as a whole. Since the instability index of gp120 amino acid sequence is less than 40, the protein tends to be stable. In addition, according to the total average value of aliphatic index and hydrophobicity, the aliphatic amino acid sequence content of gp120 amino acid sequence is relatively high and has strong hydrophilicity. It has been shown that the region of the gp41-gp120 interaction is concentrated on the side of the gp120 amino acid sequence near the N-terminus (Ref). We obtained 10 models of gp41 gp120 interactions by ClusPro 2.0: protein-protein docking server, and by analyzing the polarity of the models, we found that the regions of gp41-gp120 interactions are also mainly concentrated on the N-terminal side of the amino acid sequence. Meanwhile, the amino acid residues (Cln 130 and Asn-45) that determine the interaction between gp41 and gp120 by inducing mutations were found in our model obtained through ClusPro 2.0: protein-protein docking server (Ref.) .In the docking results, we found that the gp41-gp120 interaction sites are mainly composed of hydrophobic amino acids and charged amino acids, and these amino acid residues interact with each other through hydrogen bonds and electrostatic gravitation.

JP conceived and designed the research; JP, LY, HF, ZX and XL wrote the manuscript; LY, HF, ZX and PP performed experiments; LY, HF, ZX and PP analyzed data. JP reviewed the manuscript.

This study was supported by research grants, Academic Enhancement Support Program of Hainan Medical University (Grant No: XSTS2025174)

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