Structures of the ribosome bound to EF-Tu-isoleucine tRNA elucidate the mechanism of AUG avoidance.

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.

The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs.

Leucine and Isoleucine are two amino acids that differ only by the positioning of one methyl group. This small difference can have important consequences in α-helices, as the ß-branching of Ile results in helix destabilization. We set out to investigate whether there are general trends for the occurrences of Leu and Ile residues in the structures and sequences of class A GPCRs (G protein-coupled receptors). GPCRs are integral membrane proteins in which α-helices span the plasma membrane seven times and which play a crucial role in signal transmission. We found that Leu side chains are generally more exposed at the protein surface than Ile side chains. We explored whether this difference might be attributed to different functions of the two amino acids and tested if Leu tunes the hydrophobicity of the transmembrane domain based on the Wimley-White whole-residue hydrophobicity scales. Leu content decreases the variation in hydropathy between receptors and correlates with the non-Leu receptor hydropathy. Both measures indicate that hydropathy is tuned by Leu. To test this idea further, we generated protein sequences with random amino acid compositions using a simple numerical model, in which hydropathy was tuned by adjusting the number of Leu residues. The model was able to replicate the observations made with class A GPCR sequences. We speculate that the hydropathy of transmembrane domains of class A GPCRs is tuned by Leu (and to some lesser degree by Lys and Val) to facilitate correct insertion into membranes and/or to stably anchor the receptors within membranes.

Impact of non-proteinogenic amino acid norvaline and proteinogenic valine misincorporation on a secondary structure of a model peptide.

Norvaline is a straight-chain, hydrophobic, non-proteinogenic amino acid, isomeric with valine. Both amino acids can be misincorporated into proteins at isoleucine positions by isoleucyl-tRNA synthetase when the mechanisms of translation fidelity are impaired. Our previous study showed that the proteome-wide substitution of isoleucine with norvaline resulted in higher toxicity in comparison to the proteome-wide substitution of isoleucine with valine. Although mistranslated proteins/peptides are considered to have non-native structures responsible for their toxicity, the observed difference in protein stability between norvaline and valine misincorporation has not yet been fully understood. To examine the observed effect, we chose the model peptide with three isoleucines in the native structure, introduced selected amino acids at isoleucine positions and applied molecular dynamics simulations at different temperatures. The obtained results showed that norvaline has the highest destructive effect on the ß-sheet structure and suggested that the higher toxicity of norvaline over valine is predominantly due to the misincorporation within the ß-sheet secondary elements.

DR7dA, a Novel Antioxidant Peptide Analog, Demonstrates Antifibrotic Activity in Pulmonary Fibrosis In Vivo and In Vitro.

Pulmonary fibrosis (PF), which is characterized by enhanced extracellular matrix (ECM) deposition, is an interstitial lung disease that lacks an ideal clinical treatment strategy. It has an extremely poor prognosis, with an average survival of 3-5 years after diagnosis. Our previous studies have shown that the antioxidant peptide DR8 (DHNNPQIR-NH2), which is extracted and purified from rapeseed, can alleviate PF and renal fibrosis. However, natural peptides are easily degraded by proteases in vivo, which limits their potency. We have since synthesized a series of DR8 analogs based on amino acid scanning substitution. DR7dA [DHNNPQ (D-alanine) R-NH2] is an analog of DR8 in which L-isoleucine (L-Ile) is replaced with D-alanine (D-Ala), and its half-life is better than that of DR8. In the current study, we verified that DR7dA ameliorated tumor growth factor (TGF)-ß1-induced fibrogenesis and bleomycin-induced PF. The results indicated that DR7dA reduced the protein and mRNA levels of TGF-ß1 target genes in TGF-ß1-induced models. Surprisingly, DR7dA blocked fibrosis in a lower concentration range than DR8 in cells. In addition, DR7dA ameliorated tissue pathologic changes and ECM accumulation in mice. BLM caused severe oxidative damage, but administration of DR7dA reduced oxidative stress and restored antioxidant defense. Mechanistic studies suggested that DR7dA inhibits ERK, P38, and JNK phosphorylation in vivo and in vitro All results indicated that DR7dA attenuated PF by inhibiting ECM deposition and oxidative stress via blockade of the mitogen-activated protein kinase (MAPK) pathway. Hence, compared with its parent peptide, DR7dA has higher druggability and could be a candidate compound for PF treatment in the future. SIGNIFICANCE STATEMENT: In order to improve druggability of DR8, we investigated the structure-activity relationship of it and replaced the L-isoleucine with D-alanine. We found that the stability and antifibrotic activity of DR7dA were significantly improved than DR8, as well as DR7dA significantly attenuated tumor growth factor (TGF)-ß1-induced fibrogenesis and ameliorated bleomycin-induced fibrosis by inhibiting extracellular matrix deposition and oxidative stress via blockade of the MAPK pathway, suggesting DR7dA may be a promising candidate compound for the treatment of PF.

Domino Effect in Allosteric Signaling of Peptide Binding.

While being a thoroughly studied model of dynamic allostery in a small protein, the pathway of signal transduction in the PDZ3 domain has not been fully determined. Here, we investigate peptide binding to the PDZ3 domain by conventional and fully data-driven analyses of molecular dynamics simulations. First, we identify isoleucine 37 as a key residue by widely used computational procedures such as cross-correlation and community network analysis. Simulations of the Ile37Ala mutant show disruption of the coordinated movements of spatially close regular elements of secondary structure. Then, we employ a recently developed unsupervised, data-driven procedure to determine an optimized reaction coordinate (slowest-relaxation eigenvector) of peptide binding. We use this reaction coordinate to improve sampling by restarting additional simulations from the transition state region. Significant differences in the distributions of some of the pairwise residue distances in the bound and unbound states emerge from the projection onto the optimized reaction coordinate. The unsupervised analysis shows that allosteric signaling is transduced from the ß2 strand, which forms part of the peptide binding site, to the spatially adjacent ß3 and ß4 strands, and from there to the α3 helix. The domino-like transmission of a (peptide binding) signal along ß strands and α helices that are close in three-dimensional space is likely to be a general mechanism of allostery in single-domain proteins.

Importance of Ile71 in ß-actin on histidine methyltransferase SETD3 catalysis.

SETD3-catalysed N3-methylation of His73 in ß-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in ß-actin on human SETD3 catalysis. Substitution of Ile71 in ß-actin peptides by its natural and unnatural mimics reveals that the 'secondary' Ile71 binding pocket modulates the substrate efficiency of ß-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-ßA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.

Study elastic properties of the leucine and isoleuicine from first principles calculations.

I studied the elastic properties of crystalline L- and DL-forms of leucine and isoleucine within the framework of density functional theory with van der Waals interactions. The energy gaps of the considered crystals are 7.48-7.60 eV. Chiral molecules have the same chemical composition. Therefore, the study of crystalline amino acids provides a better understanding of how the structure of molecules affects mechanical properties of molecular crystals. Complete set of elastic constants for L-leucine, L-isoleucine, DL-leucine and DL-isoleucine were calculated. Linear compressibility of crystals has high anisotropy. The crystalline L- and DL-forms of leucine and isoleucine have different mechanical properties. Linear compressibility has a negative value for DL-isoleucine. My calculations predict that L-leucine and L-isoleucine are ductile compounds, while DL-leucine and DL-isoleucine are brittle compounds.

Differentiation of leucine and isoleucine residues in peptides using charge transfer dissociation mass spectrometry (CTD-MS).

RATIONALE: The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations. METHODS: Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument. RESULTS: CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time. When present, d ions are typically greater than 10% of the abundance of the corresponding a ions from which they derive, and w ions are typically more abundant than the z ions from which they derive. CONCLUSIONS: CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high-energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.

Determination of modification degree of polysialylated therapeutic proteins using 1H-NMR spectroscopy.

Water soluble polymers and their derivatives bound to proteins can dramatically favor the biological activity of new drugs and vaccines. Quantification of the modification degree of the protein is crucial during the development and licensing phase and later in order to monitor the industrial production process and to match product specification. In this work, we describe an innovative way to measure directly the modification degree of polysialylated proteins using proton NMR (Nuclear Magnetic Resonance) spectroscopy. Following a calibration step, the modification degree can be easily deduced by the integration ratio of a separate signal from the polymer and selected signals from the protein. In fact, the upfield-shifted signals of methyl groups from Valine, Leucine and Isoleucine can be used as an internal calibration reference for the integration. In this paper recombinant factor VIII (rFVIII) and recombinant factor IX (rFIX) proteins modified by polysialic acid (PSA) are used to illustrate the accuracy, reproducibility and ease of the method that may replace or complement wet-chemistry approaches.

Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence of L-tryptophan.

The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand L-tryptophan (L-Trp) indicate that L-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with L-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of L-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis.

Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein.

Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T1) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of a molecule in solution masks some of these motions. Therefore, we investigate to what extent relaxometry improves timescale resolution, using the "detector" analysis of dynamics. Here, we demonstrate improvements in the characterization of internal dynamics of methyl-bearing side chains by carbon-13 relaxometry in the small protein ubiquitin. We show that relaxometry data leads to better information about nanosecond motions as compared to high-field relaxation data only. Our calculations show that gains from relaxometry are greater with increasing correlation time of rotational diffusion.

Residue Interaction Network Analysis Predicts a Val24-Ile31 Interaction May be Involved in Preventing Amyloid-Beta (1-42) Primary Nucleation.

Alzheimer's disease (AD) patients could benefit from a more effective treatment than the current FDA-approved options. Because amyloid-beta (Aß) is thought to play a central role in AD pathogenesis, many experimental drugs attempt to reduce Aß-induced pathology. Preventing amyloid accumulation may be a more effective strategy than clearing Aß plaques after they form. If preventing Aß accumulation can treat or prevent AD, then understanding Aß primary nucleation may aid rational drug design. This study examines Aß residue interaction networks and reports network and structural observations that may provide insight into primary nucleation. While many studies identify structural features of Aß that promote aggregation, this study reports features that may resist primary nucleation by examining Aß42 studies in more and less polar solvents. In Aß42 in a less polar solvent (PDB ID: 1IYT), Val24 and Ile31 have higher betweenness and residue centrality values. This may be due to a predicted interaction between Val24 and Ile31. Residues in the central hydrophobic cluster (CHC) of Aß40 and Aß42 had significantly higher betweenness values compared to the average betweenness of the structures, highlighting the CHC's reported role in oligomerization. The predicted interaction between Val24 and Ile31 may reduce the likelihood of primary nucleation of Aß.

Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity.

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.

Molecular dynamic simulations, GIAO-NMR and TD-DFT spectroscopy analyze for zwitterionic isoleucine (ILE)N , 1 ≤ N ≤ 6, in water solution.

In this article, we investigate the effects of the isoleucine (ILE)N amino acid chain growth, N = 1.0.6, the ILE conformational effect as well as the solvent presence on the electrical and magnetic spectroscopic properties when these compounds are in aqueous solution. Computational molecular dynamics simulations were performed to include the solvent medium and generate uncorrelated configurations involving solute-solvent structures. The charge point model for solvent was used to obtain the results for quantum mechanical calculation, in special DFT calculations, for (ILE)N structures. Our results for the magnetic shielding constant obtained via GIAO-DFT-NMR calculations show that there is evidence of a magnetic behavior that characterizes the number of peptide bonds and, therefore, how the N isoleucine polypeptide chain is composed. TD-DFT results also show an absorption band shift to larger wavelengths indicating a dependence on N growth.

5-Aryl-furan derivatives bearing a phenylalanine- or isoleucine-derived rhodanine moiety as potential PTP1B inhibitors.

Two series of 5-aryl-furan derivatives bearing a phenylalanine- or isoleucine-derived rhodanine moiety were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, 5g was found to have the best PTP1B inhibitory potency (IC50 = 2.66 ± 0.16 µM) and the best cell division cycle 25 homolog B (CDC25B) inhibitory potency (IC50 = 0.25 ± 0.02 µM). Enzymatic data together with molecular modeling results demonstrated that the introduction of a sec-butyl group at the 2-position of the carboxyl group remarkably improved the PTP1B inhibitory activity.

Sequence-Dependent Nanofiber Structures of Phenylalanine and Isoleucine Tripeptides.

The molecular design of short peptides to achieve a tailor-made functional architecture has attracted attention during the past decade but remains challenging as a result of insufficient understanding of the relationship between peptide sequence and assembled supramolecular structures. We report a hybrid-resolution model to computationally explore the sequence-structure relationship of self-assembly for tripeptides containing only phenylalanine and isoleucine. We found that all these tripeptides have a tendency to assemble into nanofibers composed of laterally associated filaments. Molecular arrangements within the assemblies are diverse and vary depending on the sequences. This structural diversity originates from (1) distinct conformations of peptide building blocks that lead to different surface geometries of the filaments and (2) unique sidechain arrangements at the filament interfaces for each sequence. Many conformations are available for tripeptides in solution, but only an extended ß-strand and another resembling a right-handed turn are observed in assemblies. It was found that the sequence dependence of these conformations and the packing of resulting filaments are determined by multiple competing noncovalent forces, with hydrophobic interactions involving Phe being particularly important. The sequence pattern for each type of assembly conformation and packing has been identified. These results highlight the importance of the interplay between conformation, molecular packing, and sequences for determining detailed nanostructures of peptides and provide a detailed insight to support a more precise design of peptide-based nanomaterials.

Potent cyclometallated Pd(II) antitumor complexes bearing α-amino acids: synthesis, structural characterization, DNA/BSA binding, cytotoxicity and molecular dynamics simulation.

A rational approach was adopted to design high-potential metal-based antitumor agents. A series of organometallic Pd(ii) complexes with a general formula of [Pd{κ2(C,C)-[(C6H4-2)PPh2]CH(CO)C6H4Ph-4}{κ2(N,O)}] (N,O = alanine (Pd-A), valine (Pd-V), leucine (Pd-L), l-isoleucine (Pd-I) and phenylalanine (Pd-F)) were prepared by cyclopalladation of the phosphorus ylide, bridge cleavage reaction and subsequent chelation of natural α-amino acids. The complexes were fully identified using IR and multinuclear 1H, 13C, 31P NMR spectroscopic methods. X-ray crystallography exhibited that the Pd(ii) atom is located in a slightly distorted square-planar environment surrounded by C,C-orthometallated phosphorus ylide as well as NO-pendant amino acid functionality. In vitro cytotoxicity evaluation of new cyclometallated Pd(ii) complexes toward a human leukemia (K562) cancer cell line indicated promising results. The highest cytotoxic activity was discovered in the case of phenylalanine (CH2C6H5). IC50 values of this complex on a panel of human tumor cell lines representative of liver (HepG2), breast (SKBR-3), and ovarian (A2780-Resistance/Sensitive) cancers also indicated promising antitumor effects in comparison with standard cisplatin. The binding interaction ability of the phenylalanine-containing orthopalladated complex, as the most efficient compound, with calf-thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) was investigated. UV-Vis spectroscopy, competitive emission titration, and circular dichroism (CD) techniques demonstrated the intercalative binding of the Pd(ii) complex with DNA. Molecular docking studies also fully agreed with the experimental data. Examination of the reactivity towards the protein BSA revealed that the static quenching mechanism of BSA intrinsic fluorescence by the Pd(ii) complex with a binding constant (Kb) of ∼105 is indicative of the high affinity of the complex. The competitive binding experiment using site markers with definite binding sites demonstrated that the hydrophobic cavities of site I (subdomain IIA) are responsible for the bimolecular interaction between protein BSA and the complex. Molecular docking studies effectively confirmed the significance of hydrophobic interactions in Pd(ii)-BSA binding. The results of this study could greatly contribute to exploring new potent metal-based anticancer drugs.