htf and capsaicin were purchased from Sigma Aldrich (St. Loius, United States). We prepared a stock solution of htf and it was subjected to further dilution for working concentrations. All the chemicals used for buffer preparation were obtained from HiMedia. We used quartz cuvettes for spectroscopic assays.

Molecular docking was employed to explore the binding affinity and interaction between htf and capsaicin. The structure of htf (PDB accession: 3V83) was extracted from the RCSB Protein Data Bank with. The ligand structure was retrieved from the PubChem database with CID: 1548943. The structures were processed utilizing MGL AutoDock tools (Huey et al., 2012). Molecular docking was carried out using InstaDock v1.2 to investigate detailed binding of capsaicin with htf. Htf (Mohammad et al., 2021). We used a blind search space strategy to ensure thorough exploration of potential binding sites within the protein. was employed. For the experiment, the grid coordinates were set at 82 Å for the X-axis, 99 Å for the Y-axis, and 84 Å for the Z-axis, with the central coordinates defined as follows: −52.355 along the X-axis, 17.601 along the Y-axis, and −30.21 along the Z-axis. We used a grid spacing of 1 Å and retorted employed Discovery Studio Visualizer and PyMol to carry out post docking visualization.

Molecular dynamic (MD) simulation studies were performed under a constant temperature of 300 K, using the “charmm36-jul2022” force field within the “GROMACS suite version 2020-β” (Van Der Spoel et al., 2005). The best pose that was obtained from docking studies was used as a starting structure. Ligand topology was created through the CHARMM CGenFF program. Subsequently, the integration of the ligand topology with the initial coordinates of htf was carried out using CGenFF Python script to ensure proper configuration of the system. After that the solvent molecules were introduced in the system by utilizing a 1 nm cubic box and the solvation was executed using the gmx solvate command, employing SPC216 solvent in GROMACS (Glättli et al., 2002). Post that energy minimization was carried out using the steepest descent approach to refine the systems. The resulting trajectories were investigated using various GROMACS modules, including gmx rms, gmx rmsf, gmx gyrate, gmx sasa, gmx sham, etc. The graphs and visual representations were generated using QtGrace (Turner, 2005).

We employed fluorescence binding study to check the actual binding of capsaicin with htf in line with earlier studies on Shimadzu Spectrofluorophotometer (RF-6000). The concentration of htf was kept constant (4 µM) and capsaicin was varied in the range of 0–6 µM. The data obtained was put into Modified Stern–Volmer (MSV) equation as per earlier literature to obtain the binding constant (K) of Htf-capsaicin complex (Shamsi et al., 2020). We carried out the experiment in triplicates and mean value was taken into account. All the spectra reported here are the subtracted spectra after considering the fluorescence of capsaicin by taking a blank spectra of the ligand.

The outcomes derived from our molecular docking study have uncovered a robust interaction between capsaicin and htf, emphasizing it to be a potential binding partner. This docking analysis has furnished better comprehension of the binding mode of capsaicin with htf, allowing for the recognition of critical residues involved in this interaction. The computed affinity for the htf-capsaicin was found to be −5.9 kcal/mol, accompanied by a ligand efficiency of 0.2682 kcal/mol per non-hydrogen atom. Building upon the observed binding affinity, we conducted an exhaustive analysis to explore the potential interactions stabilizing the htf-capsaicin complex. This analysis has provided a clear understanding of capsaicin’s binding mechanism against htf, highlighting its preference for occupying htf’s iron-binding pocket, strategically positioned within the deep cavity of htf (Figure 1A). This positioning establishes significant connections with residues located in the binding pocket, as depicted in Figure 1B. Notably, three hydrogen bonds are formed with Glu442, Ala443, and Gly444, crucial residues in close proximity to htf’s iron-binding site (Noinaj et al., 2012). Visual representations of the surface further accentuate capsaicin’s presence within htf’s internal cavity, as shown in Figure 1B. Moreover, a range of significant interactions, including the iron-binding site Tyr445, contributes to the stability of the protein-ligand complex, as illustrated in Figure 1C. Earlier studies have similarly reported binding of iron in vicinity of these crucial residues (Noinaj et al., 2012). Therefore, a reasonable hypothesis can be proposed that capsaicin docks in close proximity to htf’s iron-binding pocket, establishing stable interactions with various binding site residues within htf.

MD simulation is extensively utilized to explore the dynamic motions and conformational changes of macromolecules over a defined time frame. Here, we performed thorough MD simulations, spanning a duration of 200 ns, for htf and htf-capsaicin complex. Our primary aim was to explore conformational alterations, system stability, and the dynamics of interaction involved in the binding of capsaicin with htf. To assess structural dynamics, we employed the commonly used metric known as the root mean square deviation (RMSD) (Maruyama et al., 2023). The RMSD values for htf and htf-capsaicin complex did not reveal significant differences, suggesting that the capsaicin binding stabilizes htf without affecting its native conformation (Figure 2A). However, there were some initial fluctuations in RMSD in the first frame of 50 ns. The observed fluctuations can reasonably be attributed to the initial orientation of capsaicin within htf’s binding pocket. Subsequent to this initial phase, the RMSD of the htf-capsaicin complex stabilizes, emphasizing the sustained stability of the protein-ligand complex (Figure 2A).

We assessed local structural characteristics and the flexibility of each residue in the protein structure by conducting root-mean-square fluctuation (RMSF) calculations for free htf and capsaicin bound htf its (Figure 2B). The obtained RMSF plots show that there are variations in residue flexibility across htf. RMSF of htf persists fairly constant upon the binding of capsaicin, in a way showing intermittent fluctuations and stabilization peaks throughout the structure. Upon capsaicin binding, there was an elevation in residue fluctuations, specifically in the “N-terminal region,” where capsaicin is not bound. The occurrence of this phenomenon is attributable to the residual vibrations persisting throughout the course of simulation.

The radius of gyration (R _g) is a vital parameter that demonstrates protein’s conformational structure (Lobanov et al., 2008). The average R _g for htf and the htf-capsaicin complex was between 2.9 nm and 3.0 nm. The value of R _g for the htf-capsaicin complex was slightly lower in comparison to free htf and this was constant throughout the simulation (Figure 2C). The R _g plots depicted modest shifts with no major structural rearrangements in htf post capsaicin binding. This implies that binding of capsaicin induced higher stability in the native conformation of htf.

Additionally, Solvent-accessible surface area (SASA) was calculated for protein alone and in complex with ligand. SASA sheds light on the extent of a protein’s surface engagement with the surrounding solvent environment (Marsh and Teichmann, 2011). The calculated SASA values for protein alone and in complex with htf bound capsaicin ranged from 295 nm² to 315 nm², respectively. A subtle reduction in SASA was observed implying that there is a degree of conformational stability in htf upon capsaicin binding (Figure 2D). This marginal decline in SASA implies conformational stability in the htf resulting from capsaicin binding, i.e., ligand occupies some intramolecular space of the protein.

Integral hydrogen bonding plays a pivotal role in maintaining protein stability and can introduce directionality to interactions between proteins and ligands (Yunta, 2017). In the context of this study, we conducted an exhaustive examination of the stability of htf and htf-capsaicin complex. This investigation encompassed the quantification of hydrogen bonds formed within the protein structure throughout the simulation duration, as depicted in Figure 3A. Significantly, a subtle reduction in the number of hydrogen bonds becomes apparent upon the binding of capsaicin. In contrast, htf and capsaicin consistently maintaining 2–3 intermolecular hydrogen bonds throughout the simulation, as depicted in Figure 3B. Within htf’s binding pocket, the interaction with capsaicin was noted to involve 7–8 hydrogen bonds with increased fluctuations and 2–3 hydrogen bonds up to 130 ns, followed by 4–6 hydrogen bonds after 130–200 ns, demonstrating relatively enhanced stability. A detailed examination of hydrogen bonding patterns facilitates a deeper understanding of the stability of the interaction within the htf-capsaicin complex.

Proteins function through intricate atomic movements, and employing principal component analysis (PCA) offers a valuable approach to unraveling fundamental dynamics by pinpointing key principal motions (Stein et al., 2006). This approach has the capacity to illuminate insights into protein stability. In our exploration of the dynamic conformational behaviors of htf and the htf-capsaicin complex, we employed PCA and visually illustrated the changes in the conformation within the essential subspace (Figures 4A, B). Interestingly, with the introduction of capsaicin, htf demonstrates a cluster of stable states, suggesting a minor reduction in conformational exploration compared to its unbound state (Figure 4B). Overall, the results indicated that htf remained stable before and after capsaicin binding during the simulation trajectory.

To enhance our understanding of conformational dynamics, we conducted an in-depth exploration of the free energy landscapes (FELs) derived from the MD trajectories. Figure 5 vividly illustrates the FELs for htf and htf-capsaicin complex. In the unbound state of htf, the FEL shows a singular and consistently stable global minimum, primarily confined within a large single basin (Figure 5A). This suggests a well-defined and stable conformational state for htf when not bound to capsaicin. Upon the introduction of capsaicin, the overall conformational behavior of htf undergoes examination (Figure 5B). Strikingly, there is no significant alteration observed, indicating that capsaicin binding does not induce substantial changes in the conformational landscape of htf. The presence of capsaicin, as depicted in the FELs, expands the spectrum of conformational space discovered by htf, suggesting potential influences on its functional dynamics. This stability is crucial in understanding the molecular intricacies governing the interaction between htf and capsaicin and its potential implications for functional dynamics.

Fluorescence spectroscopy ascertains the actual binding affinity of ligand with the protein. For htf, tryptophan alone is main player in the intrinsic fluorescence; tyrosine’s fluorescence undergoes complete quenching when subjected to ionization or when in proximity to an “amino group,” a “carboxyl group,” or another tryptophan residue; phenylalanine has the lowest quantum yield. In silico observations showed the formation of stable htf-capsaicin complex. In a bid to further validate these computational observations, fluorescence binding studies were carried out. Intrinsic fluorescence of htf is very responsive to its micro-environment (Sarzehi and Chamani, 2010); any minimal changes in the microenvironment around its fluorophores results in corresponding changes in the fluorescence spectra. We excited htf at 280 nm, and it was found to show characteristic peak at around 335 nm, suggestive of the fact that htf is in its native form. The protein’s fluorescence intensity might decrease due to inner filter effect, which happens when the solution absorbs light at the excitation (λ_ex) and emission (λ_em) wavelengths and thus “inner filter’s effect” on the fluorescence intensity was adjusted as per earlier reported studies (Chi and Liu, 2011). With increasing concentrations of capsaicin, a corresponding decrease in the fluorescence intensity was observed, a phenomenon referred to as fluorescence quenching (Figure 6A). This dose dependent decrease fluorescence intensity in the presence of capsaicin is suggestive of the formation of htf-capsaicin complex, validating our in silico observations. We fitted the quenching data into MSV equation to obtain MSV plot (Figure 6B) with its intercept giving the binding constant of the complex. Capsaicin demonstrated significant binding with htf with a K of 3.99 × 10⁶ M⁻¹. The computed K value fall within the reported range for other protein-ligand complexes (Alhumaydhi et al., 2021; Anwar et al., 2021), indicating a substantial strength of interaction between capsaicin and htf. In conclusion, fluorescence spectroscopy along with molecular docking and molecular dynamics simulation approaches affirms the fact that capsaicin binds to htf with a significant affinity forming a stable htf-capsaicin complex.