In this report, using NMR and molecular modeling, we have studied the structure of lysozyme-S100A6 complex and the influence of tranilast [N-(3, 4-dimethoxycinnamoyl) anthranilic acid], an antiallergic drug which binds to lysozyme, on lysozyme-S100A6 and S100A6-RAGE complex formation and, finally, on cell proliferation. We have found that tranilast may block the S100A6-lysozyme interaction and enhance binding of S100A6 to RAGE. Using WST1 assay, we have found that lysozyme, most probably by blocking the interaction between S100A6 and RAGE, inhibits cell proliferation while tranilast may reverse this effect by binding to lysozyme. In conclusion, studies presented in this work, describing the protein-protein/-drug interactions, are of great importance for designing new therapies to treat diseases associated with cell proliferation such as cancers.

The proteins S100A9 and S100A12 are associated with the human S100 calcium-binding protein family. These proteins promote interaction with target proteins and alter their conformation when they bind to calcium ions in EF-hand motifs. The V domain of RAGE (Receptor for Advanced Glycation End products) is crucial for S100A9 binding. The binding of RAGE with S100 family proteins aids in cell proliferation. In this report, we demonstrate that S100A12 protein hinders the binding of S100A9 with the RAGE V-domain. We used fluorescence and NMR spectroscopy to analyze the interaction of S100A9 with S100A12. The binary complex models of S100A9-S100A12 were developed using data obtained from ¹H-¹⁵N HSQC NMR titrations and the HADDOCK program. We overlaid the complex models of S100A9-S100A12 with the same orientation of S100A9 and the RAGE V-domain. This complex showed that S100A12 protein blocks the interaction between S100A9 and the RAGE V-domain. It means S100A12 may be used as an antagonist for S100A9. The results could be favorable for developing anti-cancer drugs based on S100 family proteins.

The hydrophobic fusion peptide (FP), a critical component of the HIV-1 entry machinery, is located at the N terminus of the envelope (Env) gp41 subunit. The receptor-binding gp120 subunit of Env forms a heterodimer with gp41. The gp120/gp41 heterodimer assembles into a homotrimer, in which FP is accessible for antibody binding. Env conformational changes or “opening” that follow receptor binding result in FP relocating to a newly formed interprotomer pocket at the gp41-gp120 interface where it is sterically inaccessible to antibodies. The mechanistic steps connecting the entry-related transition of antibody accessible-to-inaccessible FP configurations remain unresolved. Here, using SOSIP-stabilized Env ectodomains, we visualize that the FP remains accessible for antibody binding despite substantial receptor-induced Env opening. We delineate stepwise Env opening from its closed state to a functional CD4-bound symmetrically open Env in which we show that FP was accessible for antibody binding. We define downstream re-organizations that lead to the formation of a gp120/gp41 cavity into which the FP buries to become inaccessible for antibody binding. These findings improve our understanding of HIV-1 entry and delineate the entry-related conformational trajectory of a key site of HIV vulnerability to neutralizing antibody.

Metastasis-associated S100A4 protein is a small calcium-binding protein typically overexpressed in several tumor forms, and it is widely accepted that S100A4 plays a significant role in the metastasis of cancer. Tumor suppressor p53 is one of the S100A4's main targets. Previous reports show that through p53, S100A4 regulates collagen expression and cell proliferation. When S100A4 interacts with p53, the S100A4 destabilizes wild type p53. In the current study, based on ¹H-¹⁵N HSQC NMR experiments and HADDOCK results, S100A4 interacts with the intrinsically unstructured transactivation domain (TAD) of the protein p53 and the pentamidine molecules in the presence of calcium ions. Our results suggest that the p53 TAD and pentamidine molecules share similar binding sites on the S100A4 protein. This observation indicates that a competitive binding mechanism can interfere with the binding of S100A4-p53 and increase the level of p53. Also, we compare different aspects of p53 activity in the WST-1 test using MCF 7 cells. We found that the presence of a pentamidine molecule results in higher p53 activity, which is also reflected in less cell proliferation. Collectively, our results indicate that disrupting the S100A4-p53 interaction would prevent cancer progression, and thus S100A4-p53 inhibitors provide a new avenue for cancer therapy.

HIV-1 entry to host cells is fulfilled by fusion, mediated by surface glycoprotein envelope (Env) trimers upon interaction with host receptors. The entry stoichiometry (T) defining the number of Env trimers required for fusion remains elusive. Stoichiometry was previously estimated using mathematical modeling of infectivity curves of pseudoviruses surface-decorated with heterotrimers containing wild-type and entry-deficient Env. Nevertheless, previous models rarely co-considered inter-protomer opening cooperativity (S, reflecting how CD4-induced conformational changes in one protomer affect the opening of adjacent protomers) and virion trimer number distributions, while experiments were limited to pseudoviruses. Here, we factored these two parameters into our models and included replication-competent virions. We provided simultaneous estimates of T and S under varying trimer number distributions and offered 2D stoichiometry maps for different Env strains. Our results depicted the interplay between viral infectivity and stoichiometry tuned by the number of trimers per virion. The estimates for all tested Env strains were prevalently higher (T ≥ 7 for BG505 or JR-FL, T ≥ 13 for NL4-3) than reported. A high degree of inter-protomer opening cooperativity was observed for the neutralization-sensitive NL4-3, while neutralization-resistant BG505 and JR-FL showed a low to intermediate degree. Entry stoichiometry and opening cooperativity were strikingly positive-correlated, implying tied inter-protomer and inter-Env cooperative interactions. Our findings provided an in-depth view of Env cooperativities during HIV-1 entry. IMPORTANCE: The sparsely distributed envelope (Env) trimers on the surface of HIV-1 work collaboratively to mediate viral entry into the host, the early step of infection. The number of interacting trimers with host receptors required for entry awaits elucidation. Here, we explored the cooperative interplay among and within Env trimers, shedding light on a previously overlooked dimension of HIV-1 entry. For the first time, we presented distributions of estimated parameters depicting the number of Env trimers and degrees of inter-protomer opening cooperativities using biologically relevant mathematic models combined with virion infectivity measurements. Our results demonstrated that the quantity of required functional trimers positively correlates with inter-protomer opening cooperativity, a feature conserved across various strains. Our findings underscore cooperative behavior as an inherent characteristic of Env dynamics during HIV-1 entry. These insights enhance our understanding of HIV-1 infection mechanisms and could inform strategies for developing effective inhibitors or neutralizing agents.

S100P, a small calcium-binding protein, associates with the p53 protein with micromolar affinity. It has been hypothesized that the oncogenic function of S100P may involve binding-induced inactivation of p53. We used ¹H-¹⁵N HSQC experiments and molecular modeling to study the molecular interactions between S100P and p53 in the presence and absence of pentamidine. Our experimental analysis indicates that the S100P-53 complex formation is successfully disrupted by pentamidine, since S100P shares the same binding site for p53 and pentamidine. In addition, we showed that pentamidine treatment of ZR-75-1 breast cancer cells resulted in reduced proliferation and increased p53 and p21 protein levels, indicating that pentamidine is an effective antagonist that interferes with the S100P-p53 interaction, leading to re-activation of the p53-21 pathway and inhibition of cancer cell proliferation. Collectively, our findings suggest that blocking the association between S100P and p53 by pentamidine will prevent cancer progression and, therefore, provide a new avenue for cancer therapy by targeting the S100P-p53 interaction.