Ligand Distances Research Articles

Evaluation of binding of potential ADMET/tox screened saquinavir analogues for inhibition of HIV-protease via molecular dynamics and binding free energy calculations

Developing novel drug molecules against HIV is a scientific quest necessitated by development of drug resistance against used drugs. We report comparative results of molecular dynamics simulation studies on 11 structural analogues of Saquinavir (SQV) – against HIV-protease that were earlier examined for pharmacodynamic and pharmacokinetic properties. We reported analogues S1, S5 and S8 to qualify the ADMET criterion and may be considered as potential lead molecules. In this study the designed molecules were successively docked with native HIV-protease at AutoDock. Docking scores established relative goodness of the 11 analogues against the benchmark for Saquinavir. The docked complexes were subjected to molecular dynamics simulation studies using GROMACS 4.6.2. Four parameters viz. H-bonding, RMSD, Binding energy and Protein–Ligand Distance were used for comparative analyses of the analogues relative to Saquinavir. The comparison and analysis of the results are indicative that analogues S8, S9 and S1 are promising candidates among all the analogues studied. From our earlier work and present study it is evident that among the three S8 and S1 qualify the ADMET criterion and between S1 and S8, the analogue S8 shows more target efficacy and specificity over S1 and have better molecular dynamics simulation results. Thus, of the 11 de novo Saquinavir analogues, the S8 appears to be the most promising candidate as lead molecule for HIV-protease inhibitor and is best suited for testing under biological system. Further validation of the proposed lead molecules through wet lab studies involving antiviral assays however is required. Communicated by Ramaswamy H. Sarma

Substituent-Mediated Transformation of Polynuclear Gold(I)-Sulfido Complexes—From Pentanuclear to Octadecanuclear Cluster-to-Cluster Transformation

Substituent-Mediated Transformation of Polynuclear Gold(I)-Sulfido Complexes—From Pentanuclear to Octadecanuclear Cluster-to-Cluster Transformation

DNA Origami Demonstrate the Unique Stimulatory Power of Single pMHCs as T-Cell Antigens

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering.

Spectroscopic/Bond Property Relationship in Group 11 Dihydrides via Relativistic Four-Component Methods.

Group 11 dihydrides MH2– (M = Cu, Ag, Au, Rg) have been much less studied than the corresponding MH compounds, despite having potentially several interesting applications in chemical research. In this work, their main spectroscopic constants (bond lengths, dissociation energies, and force constants) have been evaluated by means of highly accurate relativistic four-component coupled cluster (4c-CCSD(T)) calculations in combination with large basis sets. Periodic trends have been quantitatively explained by the charge-displacement/natural orbitals for chemical valence (CD-NOCV) analysis based on the four-component relativistic Dirac–Kohn–Sham method, which allows a consistent picture of the nature of the M–H bond to be obtained on going down the periodic table in terms of Dewar–Chatt–Duncanson bonding components. A strong ligand-to-metal donation drives the M–H bond and it is responsible for the heterolytic (HM···H–) dissociation energies to increase monotonically from Cu to Rg, with RgH2– showing the strongest and most covalent M–H bond. The “V”-shaped trend observed for the bond lengths, dissociation energies, and stretching frequencies can be explained in terms of relativistic effects and, in particular, of the relativistically enhanced sd hybridization occurring at the metal, which affects the metal–ligand distances in heavy transition-metal complexes. The sd hybridization is very small for Cu and Ag, whereas it becomes increasingly important for Au and Rg, being responsible for the increasing covalent character of the bond, the sizable contraction of the Au–H and Rg–H bonds, and the observed trend. This work rationalizes the spectroscopic/bond property relationship in group 11 dihydrides within highly accurate relativistic quantum chemistry methods, paving the way for their applications in chemical bond investigations involving heavy and superheavy elements.

Vibrational properties of 1D- and 3D polynuclear spin crossover Fe(II) urea-triazoles polymer chains and quantification of intrachain cooperativity

The vibrational dynamics of the iron centres in 1D and 3D spin crossover Fe(II) 4-alkyl-urea triazole chains have been investigated by synchrotron based nuclear inelastic scattering. For the 1D system, the partial density of phonon states has been modelled with density functional theory methods. Furthermore, spin dependent iron ligand distances and vibrational modes were obtained. The previously introduced intramolecular cooperativity parameter H coop (Rackwitz et al, Phys. Chem. Chem. Phys. 2013, 15, 15450) has been determined to −31 kJ mol−1 for [Fe(n-Prtrzu)3(tosylate)2] and to +27 kJ mol−1 for [Fe(n-Prtrzu)3(BF4)2]. The change of sign in H coop is in line with the incomplete and gradual character of the spin transition for the former as well as with the sharp transition for the latter reported previously (Rentschler and von Malotki, Inorg. Chem., Act. 2008, 361, 3646). This effect can be ascribed to the networks of intramolecular interactions in the second coordination sphere of the polymer chains, depending on the spin state of the iron centres. In addition, we observe a decreased coupling and coherence when comparing the system which displays a sharp spin transition to the system with an incomplete soft transition by analyzing molecular modes involving a movement of the iron centres.

Comparative analysis of radial expectation values in lanthanide compounds

A method is proposed for assessment of sets of radial expectation values 〈rk〉4f of lanthanide ions (Ln3+ = Pr3+ through Tm3+) by means of the ratios 〈rk〉4f2/R2k+1 where k = 2, 4, and 6, R is the mean Ln3+ – ligand distance. These ratios have been related to experimental Slater parameters Fk in the dielectric screening model. The Fk0 values of certain free – ions Ln IV have been predicted. The compared 〈rk〉4f sets have been those known from relativistic, Hartree – Fock, and self – consistent field computations. The lanthanide compounds are elpasolites Cs2NaLnCl6, cubic sesquioxides (C-Ln2O3, C2– and C3i – sites), trifluorides LnF3, and doped Ln3+:LaF3. The results of the comparison have been discussed, as well as the factors influencing the deviations from experimental Fk0 values of the free – ions Ln IV.

Europium-Pyridinedicarboxylate-Adenine Light-Up Fluorescence Nanoprobes for Selective Detection of Phosphate in Biological Fluids.

Phosphate (Pi) plays important roles in various physiological processes. Its quantification in biological fluids is highly crucial for timely warning of Pi accumulation. Herein, an europium (Eu)-based coordination polymer nanoprobe (Eu/DPA/Ade) is prepared by coordinating 2,6-pyridinedicarboxylic acid (2,6-DPA) and adenine (Ade) with Eu3+. Eu/DPA/Ade exhibits light-up fluorescence response to Pi. The strong coordinating interaction between Eu3+ and O atoms in the Pi group not only shortens the Eu3+-ligand distance to improve the energy transfer from 2,6-DPA to Eu3+ but also attenuates the fluorescence quenching from water molecules in the coordinating sphere of Eu3+. Eu/DPA/Ade produces red emission at λem 618 nm via the "antenna effect". The coligand Ade further promotes the fluorescent emission. The selective recognition of Pi within 10-60 μM is achieved with a detection limit of 4.65 μM. In addition, a certain level of Pi (100-170 μM) causes an exponential increment on the fluorescence of Eu/DPA/Ade and makes it feasible for visual estimation of Pi under irradiation by an ultraviolet lamp at 254 nm. The quantitative detection and visual estimation of Pi in human urine and saliva have been demonstrated.

Glass transition analysis of model metallosupramolecular polyesters bearing pendant pyridine ligands with a controlled ligand–ligand distance

Supramolecular materials formed via metal-ligand coordination, so-called metallosupramolecular materials, have attracted attention due to their ability to modify a broad range of physical properties depending on the association strength at the coordination bond. Here, we demonstrate the correlation between the glass transition properties and the ligand distance by utilizing amorphous polyesters (denoted as PE-Py) with pendant pyridine ligands arranged homogeneously along the chain. The ligand distance is treated as the pyridine group equivalent molecular weight (MPy) and takes values of 900, 750, 550, and 420. The metal salt ZnCl2 forms coordination bonds with the pyridine ligands, generating a metallosupramolecular network. The fraction of coordinated pyridine is finely tuned by the mole ratio between the pyridine ligand and ZnCl2 ([Zn2+]/[Py]) while adhering to the stoichiometric rule. DSC measurements reveal that the variation in the glass transition temperature (Tg) with increasing [Zn2+]/[Py] is closely correlated with 1/MPy. Temperature-ramp rheological measurements are also performed, revealing that the apparent activation energy (Ea) of segmental motion has a quasi-linear relationship with 1/MPy. Thus, the present study demonstrates that the degree of restriction for segmental motion is very systematically strengthened as the ligand distance decreases, highlighting the importance of ligand distance in physical property control of metallosupramolecular materials. We here demonstrate the importance of ligand–ligand distance on physical property tuning of metallosupramolecular materials by utilizing model-like polyesters bearing pyridine ligands at the side groups with a controlled ligand-distance. The ligand distance is treated as the pyridine group equivalent molecular weight (MPy) and takes values of 900, 750, 550, and 420. The metal salt ZnCl2 forms coordination bonds with the pyridine ligands, generating a metallosupramolecular network. DSC and rheological measurements revealed that the glass transition temperatures and apparent activation energy (Ea) of segmental motion changes in a close correlation with 1/MPy.

Combined Virtual Screening, DFT Calculations and Molecular Dynamics Simulations to Discovery of Potent MMP-9 Inhibitors

Background: Matrix metalloproteinase-9 (MMP-9) plays a crucial role in the development and progression of cancer. Therefore, identifying its inhibitors has enjoyed numerous attentions. In this report, a hybrid approach, including pharmacophore-based virtual screening, docking studies, and density functional theory (DFT) binding energy calculations followed by molecular dynamics simulations, was used to identify potential MMP-9 inhibitors. Methods: Pharmacophore modeling based on ARP101, as a known MMP-9 inhibitor, was performed and followed by virtual screening of ZINC database and docking studies to introduce a set of new ligands as candidates for potent inhibitors of MMP-9. The binding energies of MMP-9 and the selected ligands as well as ARP101, were estimated via the DFT energy calculations. Subsequently, molecular dynamics simulations were applied to evaluate and compare the behavior of ARP101 and the selected ligand in a dynamic environment. Results: (S,Z)-6-(((2,3-dihydro-1H-benzo[d]imidazol-2-yl)thio)methylene)-2-((4,6,7- trimethylquinazolin- 2-yl)amino)-1,4,5,6-tetrahydropyrimidin-4-ol, ZINC63611396, with the largest DFT binding energy, was selected as a proper potent MMP-9 inhibitor. Molecular dynamics simulations indicated that the new ligand was stable in the active site. Conclusion: The results of this study revealed that compared to the binding energies achieved from the docking studies, the binding energies obtained from the DFT calculations were more consistent with the intermolecular interactions. Also, the interaction between the Zinc ion and ligand, in particular the Zn2+-ligand distance, played a profound role in the quantity of DFT binding energies.

Substitution mechanisms in In-, Au-, and Cu-bearing sphalerites studied by X-ray absorption spectroscopy of synthetic compounds and natural minerals

AbstractSphalerite is the main source of In – a ‘critical’ metal widely used in high-tech electronics. In this mineral the concentration of In is commonly correlated directly with Cu content. Here we use X-ray absorption spectroscopy of synthetic compounds and natural crystals in order to investigate the substitution mechanisms in sphalerites where In is present, together with the group 11 metals. All the admixtures (Au, Cu, In) are distributed homogeneously within the sphalerite matrix, but their structural and chemical states are different. In all the samples investigated In3+replaces Zn in the structure of sphalerite. The In ligand distance increases by 0.12 Å and 0.09–0.10 Å for the 1st and 2nd coordination shells, respectively, in comparison with pure sphalerite. The In–S distance in the 3rd coordination shell is close to the one of pure sphalerite. Gold in synthetic sphalerites is coordinated with sulfur (NS= 2.4–2.5,RAu–S= 2.35 ± 0.01 Å). Our data suggest that at high Au concentrations (0.03–0.5 wt.%) the Au2S clusters predominate, with a small admixture of the Au+solid solution with an Au–S distance of 2.5 Å. Therefore, the homogeneous character of a trace-element distribution, which is commonly observed in natural sulfides, does not confirm formation of a solid solution. In contrast to Au, the presence of Cu+with In exists only in the solid-solution state, where it is tetrahedrally coordinated with S atoms at a distance of 2.30 ± 0.03 Å. The distant coordination shells of Cu are disordered. These results demonstrate that the group 11 metals (Cu, Ag and Au) can exist in sphalerite in the metastable solid-solution state. The solid solution forms at high temperature via the charge compensation scheme 2Zn2+↔Me++Me3+. The final state of the trace elements at ambient temperature is governed by the difference in ionic radii with the main component (Zn), and concentration of admixtures.

Comparative Study of Complexes of Rare Earths and Actinides with 2,6-Bis(1,2,4-triazin-3-yl)pyridine

Complexes of group III metals (rare earth and actinides) with 2,6-bis(5,6-dipropyl-1,2,4-triazin-3-yl)pyridine (BTP) have been investigated by computational (DFT) and, in limited cases, by experimental (FT-IR, X-ray) techniques with the goal of determining the characteristics of metal–ligand interactions. The DFT calculations using the M062X exchange-correlation functional revealed that metal–ligand distances correlate with the ionic radii of the metals, in agreement with available X-ray diffraction results on the Sc, Y, La, U, and Pu complexes. A related blue-shift trend could be observed in seven characteristic bands in the IR spectra associated with metal–ligand vibrations. The computations uncovered considerable charge transfer interactions, particularly in the actinide complexes, as important covalent contributions to the metal–ligand bonding. The covalent character of the metal–ligand bonds decreases in the actinides, from U to Cm.

A DFT study of the interaction between [Cd(H2O)3]2+ and monodentate O-, N-, and S-donor ligands: bond interaction analysis.

A series of B3LYP/6-311+G(d,p) calculations of the affinity of monodentate ligands for [Cd(H2O)3]2+ are performed. Three types of ligands containing O (phosphine oxide, lactam, amide, carboxylic acid, ester, ketone, aldehyde, ether, halohydrin, enol, furan), N (thiocyanate, amine, ammonia, azide), and S (thioester, thioketone, thiol, thiophene, disulfide) interacting atoms are investigated. The results show that phosphine oxide has the largest affinity for the cadmium cation due to the polarization of the P=O bond. As the P atom has a large atomic radius, the O atom can polarize the electronic cloud enhancing its amount of electronic charge and favoring the interaction with Cd2+. The affinity order found is phosphine oxide > thioester > lactam > amide > carboxylic acid > ester > thioketone > ketone > thiocyanate > amine > ammonia > aldehyde > ether > thiol > thiophene > enol > halohydrin > disulfide > azide > furan ligands. These results were also corroborated by the functional M06-2X. The electronic effects (resonance and induction) of neighboring groups of the interacting atom modulate the strength of metal-ligand binding. For almost all the O-donor ligands the electrostatic component has the same magnitude as the covalent term, while for the N- and S-donor ligands the covalent term is predominant. The polarization term accounts for twice the exchange term as part of the covalent component. The dispersion term varies less than 2kcal mol-1 for the complexes analyzed. The Pauli repulsion term is correlated with the metal ligand distance, increasing in the compounds with decreased metal-ligand bond length. The charge between the interacting atoms is also strongly correlated with both the interacting strength and the electrostatic interaction component. The natural bond orbital analysis highlights correlations of the bond order, and S and P contributions of the interacting metal-ligand orbital with the coordination strength. Graphical abstract The affinity of 20 monodentate ligands with different functional groups for the [Cd(H2O)3]2+ cation is calculated based on the interaction enthalpy and Gibbs free energy for the substitution of one water molecule from the fully hydrated cation. The affinity is correlated with geometric, electronic, and energetic parameters of the ligands and the complexes as well as with energy decomposition and natural bond order analyses results.

Analysis of paramagnetic 3d ions (Cr3+ and Fe3+) centers in fluoroelpasolite Cs2NaGaF6 crystal by both DFT and SPM calculations

The structural properties of 3d ions (Cr3+ and Fe3+) centers in Cs2NaGaF6 have been investigated by performing density functional theory (DFT) and semi-empirical superposition model (SPM) calculations. The local geometry (i.e. equilibrium ligand distance and angles between the host cation ion (Cs+, Na+, or Ga3+) and F ligands) for pure, Cr3+ doped and Fe3+ doped structures have been determined after a fully relaxed geometry optimization at each lattice site. The previous experimental zero-field splitting (ZFS) parameters (ZFSPs) have been analyzed by SPM calculations using the obtained structural properties from DFT. The combination of two methods provide us to confirm the main result of the previous electron magnetic resonance and optical investigations stating that transition metal (TM) ions substitute for octahedral sites instead of being distributed randomly in the lattice of Cs2NaGaF6.

Stable host–guest complexes of bis-2,6-pyridinedicarboxylate iron(III) with dihydroxybenzenes

The high reactivity of iron(III) complexes towards phenolic compounds, such as catechol, is well known. We show here that concealing the iron(III) site by ligands helps to obtain stable host–guest iron(III) complexes with phenolic compounds. Host–guest complexes of Htriam[Fe(2,6-pdc)2] with catechol and resorcinol (where 2,6-pdc=2,6-pyridinedicarboxylate and Htriam is mono-protonated 6-phenyl-1,3,5-triazine-2,4-diamine) were prepared and characterized. The observed color for the inclusion complex of Htriam[Fe(2,6-pdc)2] with catechol in the solid state has a distinction from the colored species generated in solution by interactions between the same components. The solid state structures of these two host–guest complexes have differences in the CO distances of dihydroxybenzene ligands as well as the arrangements of π-stacks; in the case of resorcinol, phenolic character is retained. In the catechol host–guest complex the CO distances in catechol are 1.310(7) and 1.370(6)Å, whereas in the resorcinol host–guest complex similar CO distances are 1.368(4) and 1.374(3)Å. The dissimilarity in the CO bond distances of catechol in the host–guest complex suggests charge-transfer through a semi-quinone cation radical type interactions with the iron ion showing an absorption at 570nm for the solid sample. A prior activation of catechol in the second coordination sphere through a ligand–catechol interaction is depicted.

Distance-dependent adhesion of vascular cells on biofunctionalized nanostructures

Abstract Cell-cell and cell-extracellular matrix (ECM) adhesion regulates fundamental cellular functions and is crucial for cell-material contact. Adhesion is influenced by many factors like affinity and specificity of the receptor-ligand interaction or overall ligand concentration and density. To investigate molecular details of cell-ECM and cadherins (cell-cell) interaction in vascular cells functional nanostructured surfaces were used Ligand-functionalized gold nanoparticles (AuNPs) with 6-8 nm diameter, are precisely immobilized on a surface and separated by non-adhesive regions so that individual integrins or cadherins can specifically interact with the ligands on the AuNPs. Using 40 nm and 90 nm distances between the AuNPs and functionalized either with peptide motifs of the extracellular matrix (RGD or REDV) or vascular endothelial-cadherins (VEC), the influence of distance and ligand specificity on spreading and adhesion of endothelial cells (ECs) and smooth muscle cells (SMCs) was investigated. We demonstrate that RGD-dependent adhesion of vascular cells is similar to other cell types and that the distance dependence for integrin binding to ECM-peptides is also valid for the REDV motif. VEC-ligands decrease adhesion significantly on the tested ligand distances. These results may be helpful for future improvements in vascular tissue engineering and for development of implant surfaces.

Temperature dependence of local structural changes around transition metal centers Cr3+ and Mn2+ in RAl3(BO3)4 crystals studied by EMR

Theoretical investigations are carried out to determine the temperature dependence of the local structural parameters of Cr3+ and Mn2+ ions doped into RAl3(BO3)4 (RAB, R = Y, Eu, Tm) crystals. The zero-field splitting (ZFS) parameters (ZFSPs) obtained from the spin Hamiltonian (SH) analysis of EMR (EPR) spectra serve for fine-tuning the theoretically predicted ZFSPs obtained using the semi-empirical superposition model (SPM). The SPM analysis enables to determine the local structure changes around Cr3+ and Mn2+ centers in RAB crystals and explain the observed temperature dependence of the ZFSPs. The local monoclinic C2 site symmetry of all Al sites in YAB necessitates consideration of one non-zero monoclinic ZFSP (in the Stevens notation, b21) for Cr3+ ions. However, the experimental second-rank ZFSPs (D=b20, E=1/3b22) were expressed in a nominal principal axis system. To provide additional insight into low symmetry aspects, the distortions (ligand's distances ΔRi and angular distortions Δθi) have been varied while preserving monoclinic site symmetry, in such way as to obtain the calculated values (D, E) close to the experimental ones, while keeping b21 close to zero. This procedure yields good matching of the calculated ZFSPs and the experimental ones, and enables determination of the corresponding local distortions. The present results may be useful in future studies aimed at technological applications of the Huntite-type borates with the formula RM3(BO3)4. The model parameters determined here may be utilized for ZFSP calculations for Cr3+ and Mn2+ ions at octahedral sites in single-molecule magnets and single-chain magnets.

Local tetragonal distortions of the (CrF6)3− and (FeF6)3− octahedral clusters in Cr3+- and Fe3+-doped tetragonal Rb2KGaF6 crystals

ABSTRACTThe local tetragonal distortions (α − α0) (where α is the angle defined as tgα = R⊥/R//, R⊥ and R// are the metal–ligand distances parallel with and normal to the C4 axis, α0 = 45° is the same angle in cubic symmetry) of (CrF6)3− and (FeF6)3− octahedral clusters in the tetragonal Rb2KGaF6 crystals are estimated by analyzing their electron paramagnetic resonance (EPR) zero-field splittings D. The results indicate that the two impurity octahedra and hence the host (GaF6)3− octahedra are tetragonally elongated. The distortion (α − α0) in magnitude differs from impurity to impurity because of the different sizes and natures of these impurities. These results are analogous to those in ABX3 and doped ABX3 perovskite crystals where the cubic-to-tetragonal phase transition is due to the rotation of BX6 octahedra associated with the release or elongation of B–X bond along the C4 rotational axis.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.

Using Eu3+ as an atomic probe to investigate the local environment in LaPO4–GdPO4 monazite end-members

In the present study, we have investigated the luminescent properties of Eu3+ as a dopant in a series of synthetic lanthanide phosphates from the monazite group. Systematic trends in the spectroscopic properties of Eu3+ depending on the size of the host cation and the dopant to ligand distance have been observed. Our results show that the increasing match between host and dopant radii when going from Eu3+-doped LaPO4 toward the smaller GdPO4 monazite decreases both the full width at half maximum of the Eu3+ excitation peak, as well as the 7F2/7F1 emission band intensity ratio. The decreasing Ln⋯O bond distance within the LnPO4 series causes a systematic bathochromic shift of the Eu3+ excitation peak, showing a linear dependence of both the host cation size and the Ln⋯O distance. The linear relationship can be used to predict the energy band gap for Eu3+-doped monazites for which no Eu3+ luminescent data is available. Finally, mechanisms for metal-metal energy transfer between host and dopant lanthanides have been explored based on recorded luminescence lifetime data. Luminescence lifetime data for Eu3+ incorporated in the various monazite hosts clearly indicated that the energy band gap between the guest ion emission transition and the host ion absorption transition can be correlated to the degree of quenching observed in these materials with otherwise identical geometries and chemistries.

Molecular perspective of antibody aggregates and their adsorption on Protein A resin

Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development.